There are several areas of development that are being followed in order to improve protein characterization capabilities. First, we have developed a novel approach to providing sequence information for proteins that are not described in data bases, due to data base error, incompleteness splice variants or SNPs; this incompleteness is associated most frequently with organisms having unknown or partially characterized genomes, e.g. X. laevis. We are taking the approach we have termed De Novo Peptide Sequencing through Exhaustive Enumeration of Peptide Compositions, EEPC. We have recently modified this approach using an improved algorithm that uses a graph theory approach to the solution of the problem enabling us to limit the size of the LIPCUT data base, the exhaustive listing of all peptide mass combinations, to a 500 Da range. This has increased the computational speed 10-fold. We have also extended the capability of the program to account not only for internal fragments in the observed spectra, but we have added the capability to include many post-translational modifications of the peptides being sequenced. The methodology is being evaluated independently by a laboratory at NIST.[unreadable] [unreadable] In a area related to protein identification and sequencing, we are characterizing the C-terminal post-translational modifications of tubulins. In addition to the sample preparation advances that were reported previously, in the past year we have developed a means of developing reliable spectra from multiple replicate measurements. Our approach is based on the formation of a consensus spectrum and consists of calculating a mean centered spectrum from multiple replicates. We have improved upon existing library formation methods by the recognition that a vector dot product, when used to assess similarity, can be assigned a statistical confidence at a 95% confidence level with the use of the Fisher Z-transform. The use of this approach has enabled us to make a robust comparison of tubulin isotoype expression an posttranslational modification patterns in rat and cow brain tubulin. In addition, this approach is applicable in a general sense to any mass spectral data and its use should result in the generation of more reliable spectra for a number of purposes. In this light the approach has been applied to replicate peptide fragmentation spectra and has demonstrated that its use leads to more reliable determination of de novo sequences from the resultant consensus spectrum.[unreadable] [unreadable] In an area related to characterization of protein posttranslational modifications, we have developed a methodology for the quantification of site specific tyrosine phosphorylation. Using the model system of activated high affinity IgE receptors in an electrospray ionization mass spectrometer, the quantification has been carried out using a set of external peptide standards. The binding of IgE to high affinity IgE receptors on mast cells is the initial step in activation of signaling pathways that initiate an inflammatory response. It has been demonstrated that the Src family of protein tyrosine kinases are required for this activation, however the detailed molecular mechanism has not been elucidated. The gamma subunit of the IgE receptor contains two tyrosine residues near the carboxyl terminus that are potential sites for phosphorylation. Site directed mutation of each tyrosine demonstrated that both tyrosines can be substrates for phosphorylation. Based on the phosphopeptides observed in tryptic digests, peptides were synthesized with either phospho-Tyr or Tyr at the appropriate sites. Synthetic peptides corresponding to both of the two candidate regions were used to generate standard curves to calculate relative ionization responses for phospho-Tyr and Tyr, and used to calculate the stoichiometry for phospho-Tyr at each site. Using standard curves for each pair of standards, and measuring the ratio of phosphorylated to unmodified peptide for each of the candidate tyrosine residues, it was demonstrated that Tyr65 was phosphorylated at significantly higher level than Tyr76 in the activated IgE receptor gamma subunit. While used for this specific system, clearly the approach is a general one and could be used widely to characterize phosphorylation stoichiometry.[unreadable] [unreadable] In another area, unrelated to proteins, we have been developing methodology to quantify cardiolipins in human serum by mass spectrometry. The purpose of this effort is in association with a clinical study being developed by the Institute to evaluate the effects of antibiotic treatment of pregnant women with Group B streptococcal (GBS)infections. The hypothesis of the study is that the typical peri-natal penicillin treatment gives rise to a large increase of circulating cardiolipins in the infant which then leads to respiratory distress. It has been demonstrated in other studies in fetal sheep that the GBS organisms secrete a specific cell wall membrane cardiolipin with penicillin treatment and that this substance causes respiratory distress in the sheep. It is not known whether the respiratory distress observed in a fraction of infants born to GBS colonized mothers is a result of a similar effect, or perhaps by a related effect caused by a release of endogenous cardiolipins stimulated by the bacterial death. We have developed cardiolipin extraction procedures for small quantities, ca. 20 uL, of serum in recognition of the limited sample sizes that will be available from infant cord blood at delivery. We have developed appropriate quantification standards that in conjunction with a standard additions approach will allow for quantification. In addition, we have developed a reliable method for introducing this standard into a serum sample and have shown that cardiolipin can be extracted from serum with about a 75% efficiency. At present we are refining the mass spectrometric methods for the determination of these materials.